Optical microscopy with phototransformable optical labels

Abstract

A method of imaging with an optical system characterized by a diffraction-limited resolution volume is disclosed. In a sample that includes a plurality of phototransformable optical labels (“PTOLs”) distributed in the sample with a density greater than an inverse of the diffraction-limited resolution volume of the optical system, a first subset of the PTOLs in the sample are activated, and the density of the activated PTOLs in the first subset is less than the inverse of the diffraction-limited resolution volume. A first portion of the PTOLs in the first subset of PTOLs is excited. Radiation emitted from the activated and excited PTOLs in the first portion of PTOLs is detected with the imaging optics, and locations of activated and excited PTOLs in the first portion of PTOLs is determined with a sub-diffraction-limited accuracy based on the detected radiation emitted from the activated and excited PTOLs.

1. A method of imaging with an optical system characterized by a diffraction-limited resolution volume, the method comprising:
 in a sample comprising a plurality of phototransformable optical labels (“PTOLs”) distributed in the sample with a density greater than an inverse of the diffraction-limited resolution volume of the optical system, activating a first subset of the PTOLs in the sample, wherein the density of the activated PTOLs in the first subset is less than the inverse of the diffraction-limited resolution volume; 
 exciting a first portion of the PTOLs in the first subset of PTOLs; 
 detecting radiation emitted from the activated and excited PTOLs in the first portion of PTOLs with the imaging optics; and 
 determining locations of activated and excited PTOLs in the first portion of PTOLs with a sub-diffraction-limited accuracy based on the detected radiation emitted from the activated and excited PTOLs. 
 
     
     
       2. The method of  claim 1 , wherein activating the first subset of the PTOLs comprises providing sufficient energy to the PTOLs in the first subset to transform the PTOLs from an unactivated state to an activated state. 
     
     
       3. The method of  claim 2 , wherein providing sufficient energy to the PTOLs in the first subset to transform the PTOLs from an unactivated state to an activated state comprises providing activation radiation to the sample, wherein the activating radiation has a wavelength selected to transform the PTOLs from the unactivated state to the activated state. 
     
     
       4. The method of  claim 1 , wherein exciting the first portion of PTOLs comprises providing sufficient energy to the first portion of PTOLs to excite the portion of PTOLs from a ground state to an excited state. 
     
     
       5. The method of  claim 4 , wherein providing energy to the first portion of PTOLs comprises providing excitation radiation to the sample, wherein the excitation radiation has a wavelength selected to transform the first portion of PTOLs from the ground state to the excited state. 
     
     
       6. The method of  claim 1 , further comprising generating an image based on the determined locations of the activated and excited PTOLs. 
     
     
       7. The method of  claim 1 , further comprising:
 deactivating PTOLs in the first subset of PTOLs; 
 activating a second subset of the PTOLs in the sample; 
 exciting a second portion of the PTOLs in the second subset of PTOLs, wherein a density of PTOLs in the second portion is less than the inverse of the diffraction-limited resolution volume; 
 detecting radiation emitted from the activated and excited PTOLs in the second portion of PTOLs with the imaging optics; and 
 determining locations of activated and excited PTOLs in the second portion of PTOLs with a sub-diffraction-limited accuracy based on the detected radiation emitted from the activated and excited PTOLs in the second portion of PTOLs. 
 
     
     
       8. The method of  claim 7 , wherein the first and second subsets are statistically sampled subsets of the PTOLs in the sample. 
     
     
       9. The method of  claim 7 , wherein deactivating PTOLs in the first subset of PTOLs comprises providing sufficient excitation radiation to the sample to photobleach activated PTOLs in the first subset of PTOLs. 
     
     
       10. The method of  claim 7 , wherein deactivating PTOLs in the first subset of PTOLs comprises providing resetting radiation to PTOLs in the first subset of PTOLs. 
     
     
       11. The method of  claim 7 , wherein deactivating PTOLs in the first subset of PTOLs  claim 7  comprises allowing a period of time to elapse during which PTOLs in the first subset of PTOLs decay to an unactivated state. 
     
     
       12. The method of  claim 7 , wherein deactivating PTOLs in the first subset of PTOLs occurs before activating the second subset of the PTOLs. 
     
     
       13. The method of  claim 7 , further comprising:
 recording first intensity signals of radiation emitted from PTOLs in the first portion of PTOLs as a function of a detection location; 
 analyzing the first intensity signals to determine locations of the PTOLs in the first portion of PTOLs to a sub-diffraction limited accuracy; 
 recording second intensity signals of radiation emitted from PTOLs in the second subset of PTOLs as a function of a detection location; and 
 analyzing the second intensity signals to determine locations of the PTOLs in the second portion of PTOLs to a sub-diffraction limited accuracy. 
 
     
     
       14. The method of  claim 7 , further comprising generating a sub-diffraction-limited image based on the determined locations of PTOLs in the first and second portions. 
     
     
       15. The method of  claim 7 , wherein activating the second subset of PTOLs comprises providing sufficient energy to the PTOLs in the second subset to transform the PTOLs from an unactivated state to an activated state. 
     
     
       16. The method of  claim 15 , wherein providing sufficient energy to the PTOLs comprises providing activation radiation to the sample, wherein the activating radiation has a wavelength selected to transform the PTOLs from the unactivated state to the activated state. 
     
     
       17. The method of  claim 7 , wherein exciting the second portion of PTOLs comprises providing sufficient energy to the PTOLs in the second subset to excite the second portion of PTOLs from a ground state to an excited state. 
     
     
       18. The method of  claim 17 , wherein providing energy to the PTOLs in the second subset comprises providing excitation radiation to the sample, wherein the excitation radiation has a wavelength selected to transform the PTOLs from the ground state to the excited state. 
     
     
       19. The method of  claim 7 , further comprising repeating at least twenty times the steps of:
 activating an Nth subset of the PTOLs in the sample, wherein a density of PTOLs in the Nth subset is less than the inverse of the diffraction-limited resolution volume; 
 exciting a portion of the PTOLs in the Nth subset of PTOLs; 
 detecting radiation emitted from the activated and excited PTOLs in the Nth subset of PTOLs with the imaging optics; 
 determining locations of activated and excited PTOLs in the Nth subset of PTOLs with a sub-diffraction-limited accuracy based on the detected radiation emitted from the activated and excited PTOLs in the Nth subset of PTOLs; and 
 deactivating PTOLs in the Nth subset of PTOLs, 
 wherein N is an integer that runs from 1 to 20. 
 
     
     
       20. The method of  claim 7 , further comprising repeating the steps of:
 activating an Nth subset of the PTOLs in the sample; 
 exciting a portion of the PTOLs in the Nth subset of PTOLs, wherein a density of PTOLs in the portion of PTOLs is less than the inverse of the diffraction-limited resolution volume; 
 detecting radiation emitted from the activated and excited PTOLs in the portion of PTOLs with the imaging optics; 
 determining locations of activated and excited PTOLs in the Nth subset of PTOLs with a sub-diffraction-limited accuracy based on the detected radiation emitted from the activated and excited PTOLs in the Nth subset of PTOLs; and 
 deactivating PTOLs in the Nth subset of PTOLs, 
 wherein N is an integer that runs from 1 to 20. 
 
     
     
       21. The method of  claim 7 , wherein the sample comprises a resist embedded with PTOLs wherein the embedded PTOLs have been subjected to exposure to a spatially structured beam, such that properties of the PTOL are measurably changed by such exposure, and further comprising generating an exposure profile for the resist from the determined locations of the detected PTOLs. 
     
     
       22. The method of  claim 1 , wherein the sample comprises a first species and a second species of PTOL, and further comprising:
 distinguishing the first species from the second species based on at least one of emission characteristics of the first and second species and excitation characteristics of the first and second species; and 
 determining locations of activated PTOLs in the first activated subsets for the first and second species relative to one another with sub-diffraction limited accuracy. 
 
     
     
       23. The method of  claim 1 , wherein the PTOLs comprise fluorescent proteins (“FPs”). 
     
     
       24. The method of  claim 23 , wherein the FPs comprise variants of proteins derived from the  Aequorea  genus of jellyfish by genetic modification. 
     
     
       25. The method of  claim 24 , where the variants of proteins derived from the  Aequorea  genus of jellyfish by genetic modification are selected from the group consisting of PA-GFP and PS-CFP. 
     
     
       26. The method of  claim 23 , wherein the FPs comprise variants of proteins derived any of the corals selected from the group consisting of  Discosoma striata, Trachyphyllia geoffroyi, Montastraea cavernosa, Ricordea florida, Lobophyllia hemprichii, Anemonia sulcata , and  Favia favus  by genetic modification. 
     
     
       27. The method of  claim 26 , wherein the variants of proteins derived any of the corals are selected from the group consisting of Kaede, Kikume, EosFP, and KFP. 
     
     
       28. The method of  claim 23 , wherein the FPs comprise variants of proteins derived from the Pectiniidae family of stony reef corals by genetic modification. 
     
     
       29. The method of  claim 28  wherein the variants of proteins derived from the Pectiniidae family of stony reef corals by genetic modification comprise Dronpa.